KR101055392B1 - Composition comprising the extract of halocynthia roretzi which contains antimicrobial peptide for preventing and treating gastrointestinal diseases - Google Patents

Abstract

The present invention comprises the steps of obtaining a body fluid from sea bream; Destroying the cell membrane of the bodily fluid cell separated from the bodily fluid; And a composition for the prevention and treatment of diseases of the gastrointestinal tract, which has an inhibitory activity on the growth of Helicobacter pylori containing the antimicrobial peptide-containing sesame seed extract obtained by the method comprising the step of extracting the bodily fluid cells in which the cell membrane is destroyed with an acid solvent. In detail, the antimicrobial peptide-containing sesame seed extract of the present invention can be usefully used as a pharmaceutical composition for the treatment and prevention of diseases of the gastrointestinal tract by confirming the growth inhibition activity of Helicobacter pylori.

Antibacterial Peptides, Sesame Seed Extract, Helicobacter Pylori, Gastrointestinal Disease

Description

COMPOSITION COMPRISING THE EXTRACT OF HALOCYNTHIA RORETZI WHICH CONTAINS ANTIMICROBIAL PEPTIDE FOR PREVENTING AND TREATING GASTROINTESTINAL DISEASES}

The present invention relates to a composition for the prevention and treatment of diseases of the gastrointestinal tract containing an antimicrobial peptide-containing sesame seed extract as an active ingredient.

Helicobacter pylori is a Gram-negative bacillus inhabiting human epithelial cell mucosa. It is a bacterium that grows under aerobic conditions and is isolated from the gastric mucosa in 1983. It was classified by the World Health Organization as a cause of gastric cancer. Helicobacter pylori eradication has become an essential factor for treatment in patients with peptic ulcer.

Helicobacter pylori has a worldwide infection rate of about 50%. In particular, in Korea, the infection rate is about 70-80%.

Antibiotic and proton pump inhibitor combination therapy is used for the treatment of Helicobacter pylori infection, but since it requires the use of powerful antibiotics such as amoxicillin, clerismycin, and metronidazole, the expression of antibiotic resistance and the disturbance of enteric beneficial flora by antibiotics There is a problem that appears side effects such as diarrhea caused by.

To reduce the infection rate of Helicobacter pylori, which is believed to be infected through the mouth, food, and feces of others and has a high infection rate and is directly or indirectly related to gastritis, gastric ulcer and gastric cancer worldwide, hygienic diet, clean lifestyle, In addition, aggressive Helicobacter treatment is required, and it is necessary to reduce the infection rate by proactive treatment for potential patients who are not carriers, especially mild symptoms or symptoms.

In addition, antimicrobial peptides, also known as the primitive immune system, are widely distributed from amoeba to humans, and are considered as bioprotective agents from invading pathogens, especially in invertebrate animals that do not have an immune system. It is effective for resistant pathogens because it combines to cause changes in cell membranes to kill pathogens, and it is difficult to induce resistance unless the cell membrane components of pathogens in the mechanism of action change fundamentally. Antibacterial group. While antibacterial peptides found in frogs, toads, and swine intestines are being used as new drugs to control antibiotic-resistant pathogens, they are struggling to commercialize because the antimicrobial peptides are weaker than existing antibiotics and difficult to secure in large quantities.

Sea squirt is a seafood that is farmed in large quantities in Korea, and is consumed as a food. The liquid that flows out when the sea bream is cut during such processing is mostly discarded. However, the crude extract extracted from it has the growth inhibitory activity of Escherichia coli and the major antimicrobial activity contained in the crude extract is peptide. It was revealed through Food Engineering Progress 11 (1), 54-59, and this suggests the applicability to the food business as a natural antibacterial agent. On the other hand, Korean Patent No. 10-0491423 has been shown to isolate the antimicrobial peptides that kill the MRSA and Salmonella pathogens from the natural sea urchin silk sea urchin, but so far the helicobacter pylori inhibitory activity of the crude extract or antimicrobial peptides Not known

The present inventors confirmed that the growth inhibitory effect on the Helicobacter pylori of the extract of the sea squirt containing an antimicrobial peptide.

 Accordingly, it is an object of the present invention to provide a composition for the prevention and treatment of diseases of the gastrointestinal tract, which contains an antimicrobial peptide-containing sesame seed extract having a helicobacter pylori growth inhibitory activity as an active ingredient.

In order to achieve the above object, the present invention comprises the steps of obtaining a body fluid from sea squirt; Destroying the cell membrane of the bodily fluid cell separated from the bodily fluid; And a pharmaceutical composition for the prevention and treatment of diseases of the gastrointestinal tract, which has an inhibitory activity on the growth of Helicobacter pylori containing the antimicrobial peptide-containing sesame seed extract obtained by the method comprising the step of extracting the humoral cells in which the cell membrane is destroyed with an acid solvent. to provide.

In this step, the acid solvent may comprise acetic acid, preferably 3 to 10% v / v acetic acid, more preferably 5% v / v acetic acid.

The gastrointestinal diseases include gastritis, gastric ulcer, gastric ulcer enteritis, gastric cancer, duodenal ulcer, intestinal obstruction, acute gastrointestinal bleeding, Crohn's disease or ulcerative colitis, preferably gastritis, gastric ulcer, duodenal ulcer or gastric ulcer enteritis.

In the manufacturing step, the step of obtaining the body fluid from the sea bream comprises the step of obtaining the sea bream body fluid by incision.

In the preparing step, the step of destroying the cell membrane of the bodily fluid cells separated from the bodily fluid includes centrifuging the bodily fluid to separate the bodily fluid cells, and ultrasonically treating the bodily fluid cells to destroy the cell membrane.

In the preparing step, the step of extracting with an acid solvent includes the step of taking the supernatant by centrifugation in a state in which the bodily fluid cells in which the cell membrane is destroyed are dispersed in an acid solvent.

Hereinafter, a method of obtaining an antimicrobial peptide-containing sesame seed extract of the present invention will be described in detail.

The bodily fluid obtained by washing and cutting the live sea urchin was filtered and then separated from the bodily fluid and the bodily fluid by centrifugation at a temperature of 2 ° C. to 20 ° C., preferably 4 ° C., which was 2 ° C. to 20 ° C., preferably Centrifugation at a temperature of 4 ° C. to store at −10 to −100 ° C., preferably at −70 ° C .; The bodily fluid cells isolated from the bodily fluid obtained in the above step are about 2 to 20 times the weight (kg), preferably about 10 times the acid solvent, preferably 3 to 10% v / v, more preferably 5% After dispersing in acetic acid of v / v, using a grinder, preferably an ultrasonic grinder, repeated 1 to 10 times, preferably 5 times at intervals of 1 to 30 seconds, preferably 15 seconds, the cell membrane of body fluid cells Destroying it; After further dispersing by adding an acid solvent, preferably 3 to 10% v / v, more preferably 5% v / v acetic acid to the humoral cells of the cell membrane obtained in the above step, 2 ℃ to 20 ℃ Preferably, the antimicrobial peptide-containing sesame crab of the present invention through a process comprising an extraction step of taking the supernatant obtained by centrifugation by stirring for 5 to 30 hours, preferably 24 hours at a temperature of 4 ℃ Extracts can be obtained .

The present invention provides a pharmaceutical composition for the prevention and treatment of diseases of the gastrointestinal tract containing the extract obtained by the above production method as an active ingredient.

The composition for preventing and treating gastrointestinal diseases of the present invention comprises the extract in an amount of 0.1 to 90% by weight based on the total weight of the composition. However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The composition comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents which may be used in combination with the extract, and which may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the antimicrobial peptide-containing sea bream extract of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

It is confirmed that the antimicrobial peptide-containing sesame seed extract of the present invention exhibits the growth inhibitory activity of Helicobacter pylori, and is easy to mass-produce, and in particular, it inhibits the production of resistant bacteria by using natural products having antimicrobial activity similar to that of conventional antibiotics. It can be usefully used as a pharmaceutical composition for treatment and prevention.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Reference Example 1. Measurement of Helicobacter pylori activity

Helicobacter pylori ATCC26695 and Helicobacter pylori ATCC43504 for active measurements It was used as a test bacterium. The culture of the test bacteria was carried out using agar medium containing 10% horse serum in Brucella broth and passaged at 24 hours to maintain the activity of the bacteria. Antibiotic diffusion assay was used to determine the antimicrobial activity of the samples against Helicobacter pylori. The medium used for the diffusion method was prepared by adding 0.7% agarose and 10% horse serum to Brucella broth, and suspended Helicobacter pylori strains with OD ₆₀₀ 1.0-1.2 and then added 10% horse serum to Brucella broth. A strain suspension was added at 0.2% v / v before the medium was hardened (about 40 ° C.), and then 25 ml of each petri dish was poured into a 90 mm diameter petri dish and cooled to room temperature to prepare a plate medium. After sufficiently removing the moisture of the plate medium, the sample to be tested was added to a sterilized paper disc of 6 mm in diameter, dried, and placed on the plate medium to measure the size of the clear zone generated after incubation for 3 days. All of Helicobacter pylori Strain cultures were performed at 37 ° C., 10% CO ₂ concentration in a CO ₂ incubator.

Example 1 Antibacterial Peptide-Containing Sea Urchin Extract Produce

1 kg of live mussels cultured in Chungmusan were washed with 70% ethanol, and then the inlet and outlet of the sea urchins were incised horizontally to collect 490ml of body fluid and 0.25g EDTA was added. Filtration with gauze to remove impurities and centrifugation (300 × g, 10min) at 4 ℃ separated body fluids and body fluid cells. The separated humoral cells were resuspended in 0.34M sucrose solution and centrifuged at 300C (10 x) at 4 ° C to obtain 9.7 g of humoral cells. The humoral cells were stored at -70 ° C.

5 g of humoral cells were dispersed in 50 ml of 5% v / v acetic acid, and then treated with ultrasonic crusher (Bandelin, Germany) five times at 15 second intervals to destroy the humoral cell membrane. 150 ml of 5% v / v acetic acid was added and stirred at 4 ° C. for 24 hours, followed by centrifugation (2000 × g, 25 min) to take only the supernatant, and concentrated under reduced pressure to prepare 47 mg of antimicrobial peptide-containing sea urchin extract. Used as an example sample.

The isolated antimicrobial peptide containing sea bream extract contained 4.7 mg of protein when quantified by Bicinchonic acid (BCA) analysis method (Smith, P. K., Anal. Biochem., 1985, vol. 150, 76-85).

Experimental Example 1 Antimicrobial Peptide-Containing Sea Urchin Extract Helicobacter pylori growth inhibition

The antimicrobial peptide-containing sesame seed extract obtained in Example 1 was dissolved in 0.01% acetic acid and diluted sequentially to inhibit the growth of Helicobacter pylori by the method of literature (Lehrer RI, J. Immuno. Methods, 1991, vol. 137, 167-173). Activity was measured. As a control, amoxicillin, an antibiotic known to have the highest antimicrobial activity, was used for Helicobacter pylori. Antimicrobial peptide-containing extract true sea squirt (1mg / ml 0.01% acetic acid ) and amoxicillin _{(Amoxicillin, 10mg / ml H 2} O) is Helicobacter pylori in the way shown in Example 1 was diluted to 10-fold for each 10 ^-5 ATCC26695 Growth inhibition was measured and the results are shown in Table 1 and FIG. 1.

Test substance
(Μg / paper disc) Blank
control 0.005 0.05 0.5 5 50 500 Amoxicilin - - +++ +++ ++++ ++++ ++++ (Μg / paper disc) Blank
control 0.001 0.01 0.1 One 10 50 Sea urchin extract - - - + ++ ++ +++ Clear zone mm (-: none, +:-10, ++: 10-20, +++: 20-30, ++++: 30-40, +++++: 40-50)

As a result, the antimicrobial peptide-containing sesame seed extract does not spread significantly due to the characteristics of the material that does not diffuse well, so it is reasonable to judge the activity based on the minimum concentration of growth-lowering ring production. As described above, it was confirmed that the antimicrobial peptide-containing sesame seed extract of the present invention exhibited similar antimicrobial activity as the control, and also because it uses natural products, it can be expected to exhibit an inhibitory effect such as the generation of resistant bacteria.

Experimental Example 2. Antimicrobial peptide-containing sea bream extract Anti-Helicobacter Pylori Activity Measurement

Conventional antimicrobial peptides have a mechanism of killing microorganisms directly, rather than inhibiting the growth of microorganisms like conventional antibiotics. In order to confirm whether the antimicrobial peptide-containing sesame seed extract of Example 1 exhibits a known mechanism of action against Helicobacter pylori, anti-helicobacter pylori activity measurement experiment was conducted using the method described in the literature (Jang, WS, FEMS Letters, 2002). , vol. 521, 81-86).

The microorganism used in the experiment was Helicobacter pylori ATCC43504, the initial cell count was set to 1 × 10 ⁷ CFU / ml. Protein concentration of antimicrobial peptide-containing sea bream extract was 100 ug / ml, and control was performed with 0.01% acetic acid, which is a solvent of antibacterial peptide-containing sea bream extract. Strain washing and dilution were performed using Muller-Hinton II medium without serum, 100ul of Helicobacter pylori ATCC43054 (1 × 10 ⁸ CFU / ml), 800ul of Muller-Hinton II medium, and 100ul of sea bream extract containing antibacterial peptides by concentration. To 1 ml. The mixed solution was reacted under 37 ° C. and 10% CO ₂ , and samples were taken every 10 minutes and 1 hour after the reaction. After centrifugation, the mixture was washed with Muller-Hinton II medium to stop the reaction. Plated in agar medium (containing 10% horse serum) and grown under conditions of 37 ℃, 10% CO ₂ . After 3 days of growth, the number of colonies grown in each medium was measured, and the mechanism of action of the antimicrobial peptide-containing sea urchin extract on Helicobacter pylori was observed. The results are shown in FIG. 2.

As a result, the population of Helicobacter pylori ATCC43504 was significantly reduced from 1 × 10 ⁷ CFU / ml to less than 1 × 10 ³ CFU / ml after 10 minutes of reaction with 100ug / ml antimicrobial peptide containing sea bream extract. Based on the results, the antimicrobial peptide-containing sea bream extract was found to possess the ability to kill directly rather than inhibit the growth of Helicobacter pylori.

Hereinafter, the preparation examples of the composition including the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation Example 1 Preparation of Powder

Sea urchin extract (Example 1) 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Sea urchin extract (Example 1) 20 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Sea urchin extract (Example 1) 20 mg

Crystalline Cellulose 3 mg

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Sea urchin extract (Example 1) 20 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Sea urchin extract (Example 1) 20 mg

Isomerized sugar 10 g

5 g of mannitol

Purified water quantity

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

1 is a diagram showing a result of measuring the inhibition of growth of Helicobacter pylori of the sea urchin-derived antibacterial peptide,

Figure 2 is a diagram showing the Helicobacter pylori killing effect of the sea urchin-derived antibacterial peptide.

Claims (7)

Obtaining bodily fluid from sea squirt; Destroying the cell membrane of the bodily fluid cell separated from the bodily fluid; And Helicobacter pylori growth inhibitory activity containing the antimicrobial peptide-containing sesame seed extract obtained by the method comprising the step of extracting the bodily fluid cells in which the cell membrane is destroyed as an active ingredient, Pharmaceutical composition for the prevention and treatment of gastritis, gastric ulcer, gastric ulcer enteritis, duodenal ulcer, gastric cancer or acute gastrointestinal bleeding. The method of claim 1, The acid solvent is a pharmaceutical composition comprising acetic acid. delete The method of claim 1, The step of obtaining the fluid from the sea squirt is a pharmaceutical composition comprising the step of obtaining a squeezed body fluid by dissecting the sea squirt. The method of claim 1, The step of destroying the cell membrane of the bodily fluid cells separated from the bodily fluids comprises the steps of separating the bodily fluid cells by centrifuging the bodily fluids, and the step of sonicating the bodily fluid cells to destroy the cell membranes. The method of claim 1, The extracting to the acid solvent comprises the step of taking a supernatant by centrifugation in a state in which the bodily fluid cells in which the cell membrane is destroyed are dispersed in an acid solvent. The method of claim 1, The composition is a pharmaceutical composition, characterized in that it comprises 0.1 to 90% by weight of the extract relative to the total weight of the composition.

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